WO2006052007A1 - Agent de régulation de glycémie - Google Patents

Agent de régulation de glycémie Download PDF

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Publication number
WO2006052007A1
WO2006052007A1 PCT/JP2005/021068 JP2005021068W WO2006052007A1 WO 2006052007 A1 WO2006052007 A1 WO 2006052007A1 JP 2005021068 W JP2005021068 W JP 2005021068W WO 2006052007 A1 WO2006052007 A1 WO 2006052007A1
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Prior art keywords
salt
receptor protein
protein
agent
receptor
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PCT/JP2005/021068
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English (en)
Japanese (ja)
Inventor
Fumika Inazuka
Akio Uchida
Yugo Habata
Makoto Kobayashi
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Takeda Pharmaceutical Company Limited
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Publication of WO2006052007A1 publication Critical patent/WO2006052007A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the present invention relates to a blood glucose regulator. Specifically, the present invention relates to an agent that regulates blood glucose by promoting or inhibiting glucose uptake through P 2 Y 2 receptors in skeletal muscle. Background art
  • Skeletal muscle is a high-density tissue that accounts for about 40% of body weight. It is positioned as one of the main organs of glucose metabolism. The increase in blood glucose concentration (blood glucose level) due to meals is quickly processed by increasing glucose (sugar) uptake activity in skeletal muscle due to insulin action. Therefore, glucose uptake activity in skeletal muscle is said to directly affect blood glucose level, and elucidation of its regulatory function is an important issue in diabetes research.
  • Glucose uptake in skeletal muscle is performed by a protein called glucose transporter (G 1 uTransporter: G LUT) that penetrates the cell membrane 12 times. GLUT forms a family of highly homologous proteins, and the presence of GLUT 1 to GLUT 7 has been reported.
  • G 1 uTransporter G LUT
  • G LUT 1 and GL UT 4 are functionally expressed, and G LU T 1 is normally localized to the cell membrane and is thought to be involved in basic sugar uptake.
  • G LUT 4 is usually present in the intracellular microsomal fraction, but it moves to the cell membrane (translocation) and functions to incorporate extracellular sugar into the cell. It has been. Stimulation of skeletal muscle such as insulin and muscle contraction is known to translocate GL UT 4 and increase the rate of sugar uptake (Nesheret a 1., Am. J. Physio, 24 9: 02 2 6-2 3 2, 1 9 8 5).
  • Gq signal via GPCR (G-protein-coupled receptor) promotes GLUT 4 membrane translocation and sugar uptake.
  • GPCR G-protein-coupled receptor
  • GPCR is a seven-transmembrane receptor that performs intracellular signal transduction through the activation of a coupled G protein (G protein: guaninenu c-'leotide—binding protein) and when the activity of the receptor.
  • Agonist is a physiologically active substance such as various hormones, neurotransmitters, etc.
  • G-proteins that are conjugated to receptors are activated by binding of these ligands to specific receptors.
  • the activated G protein activates further downstream signal transduction pathways and transmits information
  • G protein conjugated to GP CR is a trimeric protein and its ⁇ subunit Depending on the type of signal, the signal to be transmitted differs, and the subunit is classified into four families: G s, G i, G q, and G 12.
  • the signal transmitted to the G q subunit is downstream.
  • Phospholipase C Activated it is known to cause intracellular calcium mobilization.
  • G LUT 4 Contact Yopi PAF p 1 ate 1 et- activatingfactor
  • IAP I slet — activating protein
  • PI 3— kinase P inhibitors W rtmannin
  • PKC inhibitors PKC inhibitors
  • P 2 Y 2 is a GPCR (accession # BC 0 2 8 1 3 5) is one of the GPCR families belonging to AT P of the purine receptors P 2 Fuami Li one to ligand. In the P 2 family, an ion channel P 2 X family has also been identified.
  • the P 2 Y 2 receptor is a Gi- and G q-coupled GPCR and its ligands are UTP and ATP.
  • GP CR specifically expressed in skeletal muscle is G LU We thought it could be a signal for T4 membrane translocation and promotion of sugar uptake.
  • ⁇ 1 Expression of all GPCRs except for factoy GPCR Approximately 3 70 types of expression From the results of the analysis, GPCRs that were specifically highly expressed in human and mouse skeletal muscle were examined. As a result, P 2 Y 2 was identified as a GPCR that is highly expressed in both human and mouse skeletal muscle.
  • m 2 ⁇ expression level of ⁇ 2 ⁇ 2 receptor increases in myoblasts from myoblasts to myotube cells in C 2 C 12 cells.
  • An increase in the mRNA expression level of the P 2 Y 2 receptor accompanying this differentiation was also confirmed in rat skeletal muscle-derived cell L 6.
  • Specific and high expression of P 2 Y 2 receptor in skeletal muscle, ⁇ 2 agonists promote glucose uptake in mouse myotubes, and insulin-independent GLUT 4 by G q signal This suggests that the P 2 Y 2 receptor may be directly involved in sugar uptake in skeletal muscle.
  • G LUT 4 is strongly expressed stably expressed was the rat skeletal muscle-derived cells L 6 in [rho 2 Upsilon 2 receptor, glucose uptake by AT P is a ligand of the [rho 2 Upsilon 2 receptor
  • ATP that is, P 2 Y 2 agonist-specific promotion of sugar uptake was confirmed, and further research was conducted to complete the present invention.
  • a hypoglycemic agent comprising a compound that promotes the activity of a receptor protein or a salt thereof or a salt thereof;
  • a hypoglycemic agent comprising a compound that promotes expression of a receptor protein or a salt thereof or a salt thereof;
  • a blood glucose-elevating agent comprising a compound that inhibits the activity of a receptor protein or a salt thereof or a salt thereof;
  • a hyperglycemic agent comprising a compound or a salt thereof that inhibits expression of a gene for a P 2 Y 2 receptor protein or a salt thereof;
  • ⁇ 2 ⁇ 2 receptor protein or salt thereof is a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or a partial peptide or salt thereof Agent according to claims 1 to 8;
  • Antihyperglycemic agent comprising a polynucleotide comprising a polynucleotide encoding a receptor protein or a partial peptide thereof;
  • Prevention of diabetes, obesity or hyperlipidemia ⁇ The agent according to claim 11, which is a therapeutic agent;
  • the polynucleotide encoding the receptor protein is a polynucleotide having the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 2. 1 Agent described in 2;
  • An antihyperglycemic agent comprising a receptor protein or a partial peptide thereof or a salt thereof;
  • [1 6] ⁇ 2 ⁇ 2 A protein or its partial peptide, wherein the receptor protein or a salt thereof contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1.
  • An agent for increasing blood glucose comprising an antisense polynucleotide containing a base sequence complementary to or substantially complementary to the base sequence of a polynucleotide encoding a receptor protein or a part thereof ; [1 8] The agent according to claim 17, which is a hypoglycemia prevention / treatment agent;
  • P 2 Y 2 encoding receptor protein polynucleotide is SEQ ID NO: a nucleotide sequence the same or substantially the same base sequence represented by to 1 7 claims a polynucleotide having free 1 agent according to 8;
  • An antihyperglycemic agent comprising an antibody against a receptor protein or a salt thereof;
  • [2 2] ⁇ 2 ⁇ 2 A protein or its partial peptide or a peptide thereof, wherein the receptor protein or a salt thereof contains the amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1.
  • the agent according to claim 20 which is a salt;
  • ⁇ 2 ⁇ 2 receptor protein or a salt thereof is a skeletal muscle ⁇ 2 ⁇ 2 receptor protein or a salt thereof;
  • [2 9] ⁇ 2 ⁇ 2 A method for screening a compound that regulates blood glucose or a salt thereof, characterized by using a polynucleotide encoding a receptor protein;
  • the polynucleotide encoding the receptor protein is SEQ ID NO:
  • the screening method according to claim 29, which is a polynucleotide having the same or substantially the same nucleotide sequence as the nucleotide sequence represented by No. 2:
  • [3 1] characterized by containing a polynucleotide encoding a P 2 Y 2 receptor protein ', a kit for screening a compound that regulates blood glucose or a salt thereof;
  • ⁇ 2 ⁇ 2 which comprises using the receptor protein or a salt thereof, ⁇ 2 ⁇ 2 receptor protein or screening method Agonisu bets to salts thereof;
  • [3 4] (1) ⁇ the 2 Upsilon 2 receptor protein or a labeled ligand or a salt of a salt thereof, and when contacted with the protein or a salt thereof, (2) test compound and [rho 2 Upsilon 2 receptor When a labeled ligand of a body protein or a salt thereof or a salt thereof is contacted with the protein or a salt thereof,
  • the screening method according to claim 32 or 33 wherein the amount of binding between the labeled ligand of the 2 2 receptor protein or a salt thereof or a salt thereof and the protein or the salt thereof is measured. ;
  • [3 5] (1) ⁇ the 2 Upsilon 2 receptor protein or a labeled ligand or a salt of a salt thereof, and when contacted with a cell or a cell membrane fraction containing the protein, (2) test compound the and [rho 2 Upsilon 2 receptor protein or a labeled ligand or a salt of a salt thereof, in the case also cells containing the protein is in contact with the cell membrane fraction, [rho 2 Upsilon 2 receptor protein or its A binding amount of a ligand labeled with a salt or a salt thereof and a cell containing the protein or a cell membrane fraction thereof is measured.
  • Receptor protein or its salt ligand or its salt It is characterized by measuring a cell stimulating activity via a P 2 Y 2 receptor protein when a test compound is brought into contact with a cell containing the protein or its cell membrane fraction in the presence or absence.
  • [4 1] A method for lowering blood glucose characterized by promoting the activity of P 2 Y 2 receptor protein or a salt thereof;
  • [4 2] ⁇ 2 ⁇ 2 A method for lowering blood glucose, characterized by promoting expression of a receptor protein or a salt thereof;
  • [4 5] A method for increasing blood glucose characterized by inhibiting the activity of ⁇ 2 ⁇ 2 receptor protein or a salt thereof;
  • a method for lowering blood glucose comprising administering to a mammal an effective amount of a compound or a salt thereof that promotes expression of a receptor protein or a salt thereof;
  • [5 3] ⁇ 2 ⁇ 2 A method for increasing blood glucose, comprising administering to a mammal an effective amount of a compound that inhibits the activity of a receptor protein or a salt thereof or a salt thereof;
  • [5 4] ⁇ 2 ⁇ 2 A method for increasing blood glucose, comprising administering to a mammal an effective amount of a compound or salt thereof that inhibits expression of a receptor protein or a salt thereof;
  • ⁇ 2 ⁇ 2 receptor protein or a salt thereof is according to claim 5 7, or 5 8, wherein the skeletal muscle [rho 2 Upsilon 2 receptor ⁇ white matter or a salt thereof; [60] The use according to any one of claims 57 to 59, wherein the hypoglycemic agent is a method for preventing / treating diabetes, obesity or hyperlipidemia;
  • hyperglycemic agent is a hypoglycemia prevention / treatment agent
  • FIG. 1 shows the expression of mRN ⁇ in the top 40 GP CCR species in human or mouse skeletal muscle.
  • Figure 2 represents the expression distribution of P 2Y 2 mRN ⁇ .
  • FIG. 3 represents the expression of P 2 Y 2 mRNA in 'myoblast differentiation'.
  • FIG. 4 represents sugar uptake induced by ATP in rat myoblasts L 6 -S G 4 -81.
  • the P 2 Y 2 receptor protein used in the present invention is a receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1.
  • ⁇ 2 ⁇ 2 receptor protein can be used in any cell (eg, spleen cell, nerve, etc.) of, for example, human mammals (eg, guinea pigs, rats, mice, rabbits, pigs, hidges, bushes, monkeys, etc.).
  • human mammals eg, guinea pigs, rats, mice, rabbits, pigs, hidges, bushes, monkeys, etc.
  • Cells glial cells, knee ⁇ cells, knees Langerhans islets, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fibrocytes, muscle cells, fat Cells, immune cells (eg, macrophages, T cells, B cells, natura 3 killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, cartilage Cells, bone cells, osteoblasts, osteoclasts, breast cells, hepatocytes or stromal cells, or progenitors of these cells, stem cells or cancer cells) or blood cells, or their Any tissue in which cells are present, for example, brain, brain regions (eg, olfactory bulb, cephalic nucleus, basal sphere, hippocampus, hypothalamus, hypothalamic nucleus, cerebral cortex, medulla, cerebellum, occip
  • the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 is, for example, about 85% or more, preferably 90% or more, more preferably, the amino acid sequence represented by SEQ ID NO: 1.
  • Examples include amino acid sequences having about 95% homology.
  • the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 is mouse P 2 Y 2 (P 2 U) (Gen Bank Accession No. LI 4 7 5 1; SEQ ID NO: 3), rat P 2 Y 2 (P 2 U) (Gen Bank Accession No. U 0 9 4 0 2; SEQ ID NO: 4), and the like.
  • Examples of the protein containing the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 of the present invention include an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1.
  • a protein having substantially the same activity as the protein consisting of the amino acid sequence represented by SEQ ID NO: 1 is preferred.
  • the activity of substantially the same quality examples include ligand binding activity and signal information transmission action. With substantially the same quality, their activity is sex Qualitatively the same quality. Therefore, the ligand binding activity and the activity such as Danal signal transduction are equivalent (eg, about 0.1 to 100 times, preferably about 0.5 to 2 times Q, more preferably Is preferably about 0.5 to 2 times), but the degree of these activities may be different in quantitative factors such as the molecular weight of protein.
  • Measurement of activities such as ligand binding activity and signal signal transduction can be performed according to a method known per se, for example, it can be measured according to a screening method described later.
  • the P 2 Y 2 receptor protein includes: a) 1 or 2 or more (preferably about 1 to 30 and more preferably 1 to 10) in the amino acid sequence represented by SEQ ID NO: 1. Degree, more preferably several (1-5) amino acid sequences lacking amino acids, b) 1 or more amino acid sequences represented by SEQ ID NO: 1 (preferably 1 to An amino acid sequence to which about 30 amino acids, more preferably about 1 to 10 amino acids, and more preferably several (1 to 5) amino acids are added; c) in the amino acid sequence represented by SEQ ID NO: 1 1 or 2 or more (preferably about 1 to 30 amino acids, more preferably about 1 to 10 amino acids, more preferably several (1 to 5)) amino acids substituted with other amino acids Sequence, or d) Proteins containing amino acid sequences that combine them are also used. I can.
  • the P 2 Y 2 receptor protein has a left end at the heel end (amino end) and a right end at the C end (carboxyl end) in accordance with the convention of peptide labeling.
  • R in the ester e.g., methyl, Echiru, n- propyl, isopropyl also properly n - C w alkyl group such as heptyl, if example embodiment, consequent opening pentyl, C 3 _ 8 consequent such hexyl consequent opening mouth
  • Anorekinore group for example, Fuweniru, 'alpha - C 6, such as naphthyl - 12 Ariru group, for example, Ben Phenyl, phenethyl and other phenyl C 2 alkyl groups or monomethyl naphthyl such as phenylmethyl C 7 — 14 aralkyl groups and other viva commonly used as an oral ester
  • a royloxymethyl group is used.
  • the P 2 Y 2 receptor protein has a carboxyl group (or carboxylate) other than the C-terminus
  • a protein in which the carboxyl group is amidated or esterified is also used in the present invention. 2 Included in receptor protein.
  • the ester in this case, for example, the above-mentioned C-terminal ester is used.
  • [rho 2 Upsilon 2 The receptor protein, in the protein described above, Amino group protecting groups Mechionin residues of ⁇ -terminus (e.g., C chi _ 6 such as formyl group, C 2 _ 6 Arukanoiru group such Asechiru That are protected with an acyl group, etc., a dartamyl group that is cleaved in vivo on the heel side, and a dartamine-oxidized amino acid, a substituent on the side chain of an amino acid in the molecule (for example, protection _ SH, Amino group, Lee imidazole group, fin d'groups, etc.
  • C chi _ 6 such as formyl group
  • a dartamyl group that is cleaved in vivo on the heel side and a dartamine-oxidized amino acid, a substituent on the
  • Guanijino group appropriate protecting groups (e.g., formyl group, etc. C w Ashiru group such as C 2 _ 6 Arukanoi Le group such Asechiru) Or a complex protein such as a so-called glycoprotein to which a sugar chain is bound.
  • appropriate protecting groups e.g., formyl group, etc.
  • C w Ashiru group such as C 2 _ 6 Arukanoi Le group such Asechiru
  • a complex protein such as a so-called glycoprotein to which a sugar chain is bound.
  • the partial peptide of the P 2 Y 2 receptor protein may be any partial peptide of the above-mentioned ⁇ 2 ⁇ 2 receptor protein S, for example, ⁇ 2 ⁇ 2 receptor Among protein molecules of body proteins, those that are exposed to the outside of the cell membrane and have substantially the same receptor binding activity are used.
  • the partial peptide of ⁇ 2 ⁇ 2 receptor protein having the amino acid sequence represented by SEQ ID NO: 1 is an extracellular region (hydrophilic site) in the hydrophobic plot analysis. And a peptide containing the analyzed portion. A peptide partially containing a hydrophobic site can also be used. Peptides containing individual domains can also be used, but peptides that contain multiple domains simultaneously may be used.
  • the number of amino acids in the partial peptide of the present invention is at least 20 or more, preferably 50 or more, more preferably 100 or more of the constituent amino acid sequences of the acceptor protein of the present invention described above. Peptides having the above amino acid sequence are preferred. ⁇ .
  • the partial peptide used in the present invention lacks one or more (preferably about 1 to 10, more preferably several (1 to 5)) amino acids in the above amino acid sequence.
  • one or more amino acids are added to the amino acid sequence (preferably about 1 to 20, more preferably about 1 to 10, more preferably several (1 to 5)).
  • one or more (preferably about 1 to 10, more preferably several, more preferably about 1 to 5) amino acids in the amino acid sequence are substituted with other amino acids. You can do it.
  • the C-terminus is either a carboxyl group (one COOH), canolepoxylate (one COOO—), an amide (one CONH 2 ) or an ester (one COOR). It ’s okay.
  • the partial peptide used in the present invention has a carboxyl group (or carboxylate) in addition to the C-terminus, those in which the carboxyl group is amidated or esterified are also used in the present invention. Included in peptides.
  • the ester in this case, for example, the above C-terminal ester or the like is used.
  • the amino acid group of the methionine residue at the heel end is protected with a protecting group, and the heel end side is in vivo.
  • G 1 ⁇ cleaved by cleaved by pyroglutamine oxidation, substituents on the side chains of amino acids in the molecule are protected with appropriate protecting groups, or so-called sugar peptides with sugar chains attached Also includes complex peptides.
  • the salt include physiologically acceptable salts with acids or bases, and physiologically acceptable acid addition salts are particularly preferable.
  • examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, malein). Acid, succinic acid, tartaric acid, citrate, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, malein.
  • the P 2 Y 2 receptor protein or salt thereof used in the present invention can be produced from the above-described human mammalian cells or tissues by a known method for purifying receptor protein, and will be described later. It can also be produced by culturing a transformant containing DN 2 that codes for 2 2 receptor protein. Moreover, it can also be produced according to the protein synthesis method described later or this.
  • a commercially available resin for protein synthesis can be used.
  • resins include black methyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, ⁇ ⁇ ⁇ resin, 4 -Hydroxymethylmethyl phenylamide methyl resin, polyacrylamide resin, 4_ (2 ', 4, dimethyoxyphenylhydroxymethyl) phenoxy resin, 4 (2', 4'dimethyoxyphenyru F moc (Aminoethyl) phenoxy resin and the like.
  • an amino acid having an ⁇ -amino group and a side chain functional group appropriately protected is condensed on the resin according to various known condensation methods according to the sequence of the target protein.
  • Anti At the end of the reaction, the protein is cut out from the resin and at the same time, various protecting groups are removed, and an intramolecular disulfide bond forming reaction is performed in a highly diluted solution to obtain the target protein or its amide.
  • calpositimides include DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N, mono (3-dimethylaminoprolyl) carbodiimide, and the like.
  • the ability to add a protected amino acid directly to the resin together with a racemization-suppressing additive, such as HOB t, HOOB t, or a symmetric anhydride or HOB t ester is a HOOB t ester Can be added to the resin after activation of the protected amino acid in advance.
  • the solvent used for the activation of the protected amino acid and the condensation with the resin may be appropriately selected from solvents known to be usable for the protein condensation reaction.
  • solvents known to be usable for the protein condensation reaction For example, N, N-dimethylformamide, N, N-dimethylacetamide, acid amides such as N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chlorohonolem, and trifnoreo oral ethanol Alcohols such as mono-ole, sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane and tetrahydrofuran, -tolyls such as acetonitrile and propio-tolyl, methyl acetate and ethyl acetate Esters or appropriate mixtures thereof are used.
  • the reaction temperature is appropriately selected from a range known to be used for protein bond forming reaction, and is usually selected appropriately from a range of about ⁇ 20 ° C. to 50 ° C.
  • Activated amino acid derivatives are usually used in excess of 1.5 to 4 times.
  • Examples of protecting groups for the amino group of the raw material include Z, Boc, tertiary pentinorexinoreboninore, isobonorenoreoxycarboninore, and 4-methoxypendinoreoxycanorepo.
  • Nyl C 1-1 Z, B 1-- Z, adamantyl 'oxycanolebonyl, trifnoleoloacetinore, phthaloidol, honoreminore, 2-12 trophenylsnofenenore, dipheninorephosphinothioinore, F moc Etc. are used.
  • the carboxyl group is, for example, alkyl esterified (for example, methyl, ethinole, propyl, petitenole, tert-butinole, cyclopentinole, cyclohexyl, cycloheptinole, succulent cutinore, 2-adamantinore, etc.
  • alkyl esterified for example, methyl, ethinole, propyl, petitenole, tert-butinole, cyclopentinole, cyclohexyl, cycloheptinole, succulent cutinore, 2-adamantinore, etc.
  • the hydroxyl group of selenium can be protected by, for example, esterification or etherification.
  • the group suitable for esterification include a group derived from carbonic acid such as a lower alkyl group such as acetyl group, an aryl group such as benzoyl group, a benzyloxycarbonyl group, and a ethoxycarbonyl group. Used.
  • groups suitable for etherification include a benzyl group, a tetrahydrobiranyl group, and a t-butyl group.
  • Examples of the protecting group for the phenolic hydroxyl group of tyrosine include B z 1, C l 2 — B zl, 2-nitrite benzyl, B r— Z, and tertiary butyl.
  • Examples of the protecting group for imidazole of histidine include Tos, 41-Methoxy-1, 2,3-, 6-trimethylbenzene sulphononiole, DNP, benzenoreoxymethyl, B um, B oc, T rt, F moc, etc. are used.
  • Examples of the activated carboxyl group of the raw material include ⁇ "corresponding acid anhydride, azide, active ester [alcohol (eg, pentac oral phenol, 2, 4, 5 — trichlorophenol). , 2,4-dinitrophenol, cysomethino alcohol, norani trofenore, HONB, N-hydroxy succinide, N-hydroxy phthalenolide, ester with HOB t) etc.
  • As the activated amino group for example, a corresponding phosphate amide is used.
  • Examples of methods for removing (eliminating) protecting groups include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, and anhydrous hydrogen fluoride and methanesulfone.
  • Acid treatment with acid, ⁇ rifroleoromethane sulfonic acid, trifluoroacetic acid, or a mixture of these, base treatment with diisopropylpropylamine, triethylamine, piperidine, piperazine, etc., and sodium in liquid ammonia Reduction with hum is also used.
  • the elimination reaction by the acid treatment is performed at a temperature of about 120 ° C. to 40 ° C.
  • anisole for example, anisole, phenol, thioanisole, methazolesole, norac Addition of cation scavengers such as resorenoles, dimethinolesnore, 1,4-butanedithionore, 1,2-ethanedithionore is effective.
  • cation scavengers such as resorenoles, dimethinolesnore, 1,4-butanedithionore, 1,2-ethanedithionore is effective.
  • the 2,4-dinitophenyl phenyl group used as an imidazole protecting group for histidine was removed by thiophenol treatment
  • the formyl group used as an indole protecting group for tryptophan was the above 1,2-ethanedithiol, 1, 4
  • it can also be removed by alkaline treatment with dilute sodium hydroxide solution or dilute ammonia.
  • the protection of the functional group that should not be involved in the reaction of the raw material, the protecting group, the removal of the protecting group, the activation of the functional group involved in the reaction, etc. can be appropriately selected from known groups or known means.
  • the ⁇ -stroxyl group of a carboxyl terminal amino acid is amidated to be protected, After extending the peptide (protein) chain to the desired chain length on the non-group side, remove only the N-terminal ⁇ -amino protecting group of the peptide chain and the C-terminal carboxyl protecting group.
  • the removed protein is produced, and both proteins are condensed in a mixed solvent as described above.
  • the details of the condensation reaction are the same as described above.
  • all the protecting groups are removed by the above method to obtain the desired crude protein.
  • This crude protein can be purified using various known purification means, and the main fraction can be freeze-dried to obtain an amide of the desired protein.
  • ester of a protein for example, the a-carboxyl group of the carboxy terminal amino acid is condensed with a desired alcohol to form an amino acid ester, and then the ester of the desired protein is obtained in the same manner as the protein amide. Can be obtained.
  • the partial peptide of P 2 Y 2 receptor protein or a salt thereof used in the present invention is produced according to a known peptide synthesis method or by cleaving ⁇ 2 ⁇ 2 receptor protein with an appropriate peptidase. be able to.
  • a peptide synthesis method for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That, [rho 2 Upsilon 2 receptor protein by condensing portion Bae flop tides or amino acids and the remaining portion may constitute, when the product Ru-which have a protecting group for the purposes of Bae by leaving a protective group Peptide can be manufactured. Examples of known condensation methods and protecting group removal include the methods described in a) to e) below.
  • the partial peptide of the present invention can be purified and isolated by combining ordinary purification methods, for example, solvent extraction 'distillation' color chromatography / liquid chromatography / recrystallization.
  • solvent extraction 'distillation' color chromatography / liquid chromatography / recrystallization When the partial peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method. Conversely, when it is obtained as a salt, it is released by a known method. Can be converted into a body.
  • the polynucleotide encoding the P 2 Y 2 receptor protein used in the present invention contains the base sequence encoding D 2 2 receptor protein (D 2 or RNA, preferably DNA). Anything can be used.
  • the polynucleotide is a DNA encoding a P 2 Y 2 receptor protein, an RNA such as mRNA, and may be double-stranded or single-stranded. In the case of double-stranded DNA, double-stranded DNA, double-stranded RNA or DN A: RNA hybrid may be used. In the case of a single strand, it may be a sense strand (ie, a code strand) or an antisense strand (ie, a non-coding strand).
  • Examples of the DNA encoding the P 2 Y 2 receptor protein used in the present invention include genomic DNA, genomic DNA library, cDNA derived from the cells / tissues described above, and cDNA library derived from the cells / tissues described above. Any of synthetic DNA may be used.
  • the vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Further, it can also be directly amplified by reverse transcriptase polymerase chain reaction (hereinafter abbreviated as RT-PCR method) using a total RNA or mRNA fraction prepared from the above-mentioned cell tissue.
  • RT-PCR method reverse transcriptase polymerase chain reaction
  • D ⁇ ⁇ ⁇ encoding the P 2 Y 2 receptor protein used in the present invention is, for example, DNA containing a base sequence represented by SEQ ID NO: 2 or represented by SEQ ID NO: 2.
  • a P 2 Y receptor protein consisting of an amino acid sequence represented by SEQ ID NO: 1, and a base sequence that is complementary to the base sequence and a base sequence that is hybridized under highly stringent conditions; Any DNA may be used as long as it encodes a receptor protein having substantially the same quality of activity (eg, ligand binding activity, signal signaling, etc.).
  • the DNA that can hybridize with the base sequence complementary to the base sequence represented by SEQ ID NO: 2 is, for example, about 85% or more, preferably about 90% or more, with the base sequence represented by SEQ ID NO: 2. More preferably, a DNA containing a nucleotide sequence having a homology of about 95% or more is used.
  • DNA that can be hybridized with a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 2 is mouse P 2 Y 2 (P 2 U) (Gen Bank Acession No L 1 4 7 5 1; SEQ ID NO: 5), Rat P 2 Y 2 (P 2 U) (Gen Bank Accession No. U 0 9 4 0 2; SEQ ID NO: 6) It is done.
  • Hybridization can be performed according to a known method or a method similar thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). . Moreover, when using a commercially available library, it can be carried out according to the method described in the attached instruction manual. More preferably, it can be carried out according to highly stringent conditions.
  • the high stringent conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70 ° C, preferably about 60 to 6 Indicates conditions at 5 ° C.
  • a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM
  • a temperature of about 50 to 70 ° C, preferably about 60 to 6 Indicates conditions at 5 ° C.
  • the sodium concentration is about 19 mM and the temperature is about 65 ° C.
  • D ⁇ ⁇ ⁇ encoding the human P 2 Y 2 receptor protein containing the amino acid sequence represented by SEQ ID NO: 1 is DN A containing the base sequence represented by SEQ ID NO: 2. Etc. are used.
  • the polynucleotide containing a part of the base sequence of DN ⁇ ⁇ ⁇ encoding the P 2 Y 2 receptor protein used in the present invention includes not only the DNA encoding the partial peptide of the present invention described below. Including RNA Used to mean.
  • an antisense polynucleotide capable of inhibiting the replication or expression of a P 2 Y 2 receptor protein gene is used as a clone of DN ⁇ ⁇ ⁇ encoding ⁇ 2 ⁇ 2 receptor protein. It is possible to design and synthesize based on the base sequence information obtained or determined.
  • a polynucleotide can be hybridized with the RNA of the P 2 Y 2 receptor protein gene and can inhibit the synthesis or function of the RNA, or the P 2 Y 2 receptor protein. It is possible to regulate and control the expression of P 2 Y 2 receptor protein gene through the interaction with RN ⁇ ⁇ related to.
  • Polynucleotides that can be specifically hybridized with polynucleotides that are complementary to selected sequences of ⁇ 2 ⁇ 2 receptor proteins, R ⁇ ⁇ ⁇ , and RNAs associated with P 2 Y 2 receptor proteins are are useful for modulating 'controls the expression of [rho 2 Upsilon 2 receptor protein gene in vivo Contact Yopi vitro, also useful for the treatment or diagnosis of diseases.
  • the term “corresponding” means homologous to or complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes.
  • “Corresponding” between a nucleotide, nucleotide sequence or nucleic acid and peptide (protein) usually refers to the amino acid of the peptide (protein) in the instruction derived from the sequence of the nucleotide (nucleic acid) or its complement. ing. ⁇ 2 ⁇ 2 receptor protein gene 5, end hairpin loop, 5 'end 6 — base pair' repeat, 5, end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation stop codon 3 'end untranslated region, 3' end palindromic region, opium 3, and end hairpin loop can be selected as preferred target regions, but any region within the P 2 Y 2 receptor protein gene can be selected as target. sell.
  • the relationship between the target nucleic acid and a polynucleotide that can be hybridized to be complementary to at least a part of the target region can be said to be “antisense J”.
  • Polyoxyribonucleotides containing Doxyribose, Polyribonucleotides containing D-ribose, Purine or Pyridine Other types of polynucleotides that are N-glycosides of the midine base; tides, or other polymers with non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other containing special linkages (However, the polymer contains a nucleotide having a configuration that allows attachment of a base as found in DNA or RNA).
  • RNA hybrids DNA: RNA hybrids, and unmodified polynucleotides (or unmodified oligonucleotides).
  • nucleotide-modified eg with uncharged bonds (eg methinorephosphonate, phosphotriestenole, phosphonoreamidate, carpamate, etc.), charged bond or sulfur-containing Have binding (eg phosphorothioate, phosphorodithioate, etc.), eg proteins (nucleases, nucleases' inhibitors, Toxins, antibodies, signal peptides, poly-L-lysine, etc.) and sugars (for example, monosaccharides), etc., and side-current groups (eg, acridine, psoralen, etc.) Those containing chelating compounds (eg metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those with
  • nucleoside may include those containing not only purine and pyrimidine bases but also other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, eg, one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc. Or may be converted to a functional group such as ether or amine.
  • the antisense polynucleotide (nucleic acid) of the present invention is RNA, DNA, or modified nucleic acid (RNA, DNA).
  • modified nucleic acids include, but are not limited to, nucleic acid sulfur derivatives, thiophosphate derivatives, and those that are resistant to degradation of polynucleoside amide nucleoside amides. It is not something.
  • the antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, make the antisense nucleic acid more stable in the cell, increase the cell permeability of the antisense nucleic acid, increase the affinity for the target sense strand, and if it is toxic This makes the antisense nucleic acid less toxic.
  • the antisense nucleic acid of the present invention may be altered or contain modified sugars, bases and bonds, provided in special forms such as ribosomes, microspheres, applied by gene therapy, or added.
  • it can be used in an additional form as a polycationic substance such as polylysine, which acts to neutralize the charge of the phosphate group skeleton, enhance the interaction with the cell membrane, or incorporate nucleic acid.
  • examples include lipids that increase the amount of crude water (for example, phospholipids, cholesterol, etc.).
  • Preferred lipids to be added include cholestadiol and its derivatives (eg, cholesteryl chloroformate, cholesteric acid, etc.).
  • nucleic acids include the 3 'end or 5' of nucleic acids.
  • Other groups are specifically located at the 3' or 5 'end of the nucleic acid. It is a base for capping to prevent degradation by nucleases such as exonuclease and RNase. Can be mentioned. Examples of the group for capping include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol. . .
  • the inhibitory activity of the antisense nucleic acid is examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the G protein-coupled receptor protein. Can do.
  • the nucleic acid can be applied to cells by various known methods.
  • the DN ⁇ ⁇ ⁇ encoding the partial peptide of the P 2 Y 2 receptor protein used in the present invention is any as long as it contains a base sequence encoding the partial peptide used in the present invention. May be.
  • any of genomic DNA, genomic DNA library, cDNA derived from the above-described cell 'tissue, cDNA library derived from the above-described cell / tissue, and synthetic DNA may be used.
  • the vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like.
  • it can be directly amplified by RT_PCR using a mRNA prepared from the above-mentioned cell tissue.
  • the DNA encoding the partial peptide of the P 2 Y 2 receptor protein used in the present invention is, for example, (1) a partial base of D ⁇ ⁇ having the base sequence represented by SEQ ID NO: 2 (2) having a sequence, or (2) having a base sequence that is complementary to the base sequence represented by SEQ ID NO: 2 and a base sequence that hybridizes under high stringency conditions, and represented by SEQ ID NO: 1
  • the DN ⁇ ⁇ ⁇ that has is used.
  • Examples of DNA that can be hybridized with a base sequence complementary to the base sequence represented by SEQ ID NO: 2 include, for example, about 85% or more of the base sequence represented by SEQ ID NO: 2, 'preferably about 90% Or more, more preferably about 95% DNA containing a base sequence having the above homology is used.
  • DN ⁇ ⁇ that completely encodes P 2 Y 2 receptor protein or its partial peptide can be used.
  • the hybridization method can be carried out according to the method described in Molecular Cloning, 2nd u iambrook et al., Cold Spring Harbor Lao. Press, 1989). In addition, when using a commercially available library, it can be performed according to the method described in the attached instruction manual.
  • Substitution of DNA base sequence can be done by using PCR or known kits such as Mu tan TM -super Express Km (Takara Shuzo Co., Ltd.), Mu tan TM Ichi K (Takara Shuzo Co., Ltd.), etc. It can be performed according to a known method such as DA-LAPCR method, Gapped duplex method, Kunkel method or the like, or a method analogous thereto.
  • D ⁇ ⁇ which encodes the cloned P 2 Y 2 receptor protein can be used as it is depending on the purpose, or it can be digested with a restriction enzyme or added with a linker as desired.
  • the DNA may have ATG as a translation initiation codon on the 5 ′ end side, and may have TAA, TGA, or TAG as a translation termination codon on the 3 ′ end side. These translation initiation codons and translation termination codons can also be added using an appropriate synthetic DNA adapter.
  • the expression vector for P 2 Y 2 receptor protein is, for example: (ii) excising the desired DNA fragment from DNA encoding ⁇ 2 ⁇ 2 receptor protein, and (mouth) placing the DN ⁇ fragment in an appropriate expression vector. Linked downstream of the promoter Can be manufactured.
  • the vectors include plasmids derived from E. coli (eg, p BR 3 2 2, p BR 3 25, p UC 12 2, p UC 1 3), and plasmids derived from Bacillus subtilis (eg, p UB 110, p TP 5, p C 1 9 4.), plasmids derived from yeast (eg, p SHI 9, p SH 15), pacteriophages such as ⁇ phage, animal winoles such as retro winoles, vaccinia winores
  • p A l—11, p XT l, p R c ZCMV, p R c / RSV, pc DNA l / Neo, etc. are used.
  • the promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression.
  • S Ra promoter, S V 40 promoter, L TR promoter, CMV promoter, H S V -TK promoter and the like can be mentioned.
  • CMV promoter, an SR promoter or the like it is preferable to use.
  • the host When the host is Escherichia, trp promoter, lac promoter, rec A promoter, LPL promoter, lpp promoter, etc.
  • the host When the host is Bacillus, SPO promoter, SP
  • the host When the host is an enzyme such as 02 promoter or pen P promoter, PH05 promoter, PGK promoter, GAP promoter, ADH promoter, etc. are preferable.
  • the host is an insect cell, a polyhedrin promoter, P10 promoter, etc. are preferred.
  • expression vectors include enhancers, splicing signals, poly A addition signals, selectable markers, SV 40 replication origin (hereinafter sometimes abbreviated as SV 40 ori), etc. What is contained can be used.
  • a selection marker one, for example, dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [main source preparative Rekise preparative (MTX) resistance], ampicillin resistant gene (hereinafter, the Am p r Neomycin resistance gene (hereinafter, Neo r) May be abbreviated as G 4 18 resistance).
  • the target gene when used as a selection marker using (J HO (dhfr-) cells, the target gene can also be selected by using a medium that does not contain thymidine.
  • a signal sequence is added to the N-terminal side of the receptor protein of the present invention.
  • the host is a bacterium belonging to the genus Escherichia, Pho A ⁇ signal sequence, Om p A ⁇ signal sequence, etc.
  • the host is an animal cell, such as ⁇ -amylase ⁇ signal sequence, subtilisin 'signal sequence, etc.
  • the host is yeast, MF a ⁇ signal sequence, SUC 2 ⁇ signal sequence, etc. Insulin signal sequence, ct-interferon signal sequence IJ, antibody molecule / signal sequence, etc. can be used.
  • a transformant can be produced by using the thus constructed vector containing DN ⁇ encoding the P 2 Y 2 receptor protein of the present invention.
  • the host for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, etc. are used.
  • a specific example of the genus Escherichia is Escherichia coli K 1 2 ⁇ D ⁇ 1 [Procedures ⁇ The ⁇ National ⁇ The Power Demi-'op ⁇ Sciences ⁇ (Proc. Natl. Acad. Sci.
  • Bacillus spp. include Bacillus subtilis MI 1 1 4 [gene, 2 4 ⁇ , 2 5 5 (1 9 8 3)], 2 0 7 — 2 1 'Journal of Biochemistry, 9 5 ⁇ , 8 7 (1 9 8 4)], etc. are used.
  • yeast include Saccharqmyces cerevisiae AH 2 2, AH 2 2 R- ' ⁇ A 8 7— 1 1 A, DKD— 5 D, 2 OB— 1 2, Schizosaccharomyces bomb ( Schizosaccharomyces pombe) NCYC 1 9 1 3, NCYC 2 0 3 6 and Pichia pastoris.
  • insect cells for example, when the virus is Ac NPV, larvae-derived strains of larvae (Spodoptera frugiperda cell; Sf cells), MG 1 cells derived from the midgut of Trichoplusia ni, Trichoplusia ni High Five TM Itoda vesicles derived from eggs, Hozuki moon sac from Mamestra brassicae, or cells derived from Estigmena acrea are used.
  • the virus is BmNPV, cocoon-derived cell lines (Bombyxmori N; B mN cells) are used.
  • S f cells include S f 9 cells (ATCCCRL 1711), S f 21 cells (hereinafter, Vaughn, JL et al., In Vivo, 13, 213-217, (1977). ) Etc. are used.
  • insects for example, silkworm larvae are used [Maeda et al., Nature, 3 1 5 ⁇ , 5 9 2 (1 9 8 5)].
  • animal cells examples include monkey cell CO S-7, V ero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dhfr gene-deficient Chinese hamster cell C HO (hereinafter CHO (dhfr)) Abbreviated cells), mouse L cells, mouse At T-20, mouse myeloma cells, rat GH 3, human FL cells, and the like.
  • Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).
  • a liquid medium is suitable as a medium used for the culture, including a carbon source necessary for the growth of the transformant, Nitrogen sources, inorganic substances, etc. can be included.
  • carbon sources include 'glucose, dextrin, soluble starch, sucrose, etc.
  • nitrogen sources include ammonium salts, nitrates, corn stee' liquor, peptone, casein, meat extract.
  • inorganic or organic substances and inorganic substances such as soybean meal and potato extract include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride.
  • yeast extract, vitamins, growth promoting factors and the like may be added.
  • the pH of the medium is preferably about 5-8.
  • M 9 medium containing glucose and casamino acids For example, M 9 medium containing glucose and casamino acids [Miller, Journal 'Ob'Exeper in Molecular Molecular Genes (Journal) of Experiments in Molecular Genetics), 4 3 1 — 4 3 3, Cold Spring Harbor Laboratory, New York 1 9 7 2].
  • a drug such as 3in ⁇ or rilatalylic acid can be added.
  • the culture is usually carried out at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration or agitation can be added.
  • the culture is usually carried out at about 30 to 40 ° C for about 6 to 24 hours, and if necessary, aeration or agitation can be added.
  • examples of the culture medium include, but are not limited to, Burkholder I / J, medium [Bostian, KL, et al., Proc. ⁇ Sciences ⁇ The ⁇ USA (Proc. Natl. Acad. Sci. USA), 7 7 4, 4 5 0 5 (1 9 8 0)] and SD media containing 0.5% casamino acid [ Bitter, GA et al., Proc. Natl. Acad. Sci. USA, 8 1 ⁇ , 5 3 3 0 (Proc. Natl. Acad. Sci. USA) 1 9 8 4)].
  • the pH of the medium is preferably adjusted to about 5-8. Incubate at approximately 20 ° C to 35 ° C for approximately 24 to 7 to 2 hours, with aeration and agitation as necessary.
  • the medium When cultivating a transformant whose host is an animal cell, the medium may be, for example, a MEM medium containing about 5 to 20% fetal bovine serum [Science, 1 2 2 ⁇ , 5 0 1 (1 9 5 2)], DMEM medium [Virology, 8 ⁇ , 3 9 6 (1 9 5 9)], R PM I 1 6 4 0 medium [Journal of 'The' American 'Medical' Association (The Journal of the American Medical Association) 1 9 9 ⁇ , 5 1 9 (1 9 6 7)], 1 9 9 Medium [Proceeding of the Society (Proceeding of the Society) for the Biological Medicine), 7 3 ⁇ , 1 (1 9 5 0)].
  • 11 is preferably about 6-8.
  • Cultivation is usually performed at about 30 ° C to 40 ° C for about 15 to 60 hours, with aeration and agitation as necessary.
  • the P 2 Y 2 receptor protein of the present invention can be produced in the cell, in the cell membrane or outside of the transformant.
  • [rho 2 Upsilon 2 receptor protein is extracted culture or cells or found to be used in the present invention, after cultivation, collected bacteria or cells by a known method, suspended in a suitable buffer solution, super waves, fungus body ⁇ Rui such as by lysozyme and / or freeze-thaw then disrupted cells, a method of obtaining a crude extract of more [rho 2 Upsilon 2 receptor protein to a centrifugal separation or filtration is appropriately used.
  • the buffer solution may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton (registered trademark) X-100.
  • Purification of the ⁇ 2 receptor protein contained in the culture supernatant or the extract thus obtained can be performed by appropriately combining known separation and purification methods.
  • These known separation and purification methods include mainly molecular weights such as methods that utilize solubility, such as salting-out solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyatalylamide gel electrophoresis.
  • Method using difference in charge method using difference in charge such as ion exchange chromatography, method using specific newness such as affinity chromatography, difference in hydrophobicity such as reversed-phase high-performance liquid chromatography
  • a method using the difference in isoelectric point such as isoelectric focusing.
  • the P 2 Y 2 receptor protein obtained in this way is obtained in free form, it can be converted to a salt by a known method or a method analogous thereto, and conversely In some cases, it can be converted to a free form or other salt by a known method or a method analogous thereto.
  • ⁇ 2 ⁇ 2 receptor protein produced by the recombinant may be arbitrarily modified or partially removed by allowing an appropriate protein-modifying enzyme to act before or after purification. You can also.
  • protein modifying enzymes include trypsin, chymotribsin, arginyl endopeptidase, protein kinase, and glycosidase.
  • the activity of the 2 2 receptor protein produced in this way can be measured by a binding experiment with a labeled ligand and an enzyme immunoassay using a specific antibody.
  • Antibodies against [rho 2 Upsilon 2 receptor protein used in the present invention if an antibody capable of recognizing the need is [rho 2 Upsilon 2 receptor protein use in the present invention, polyclonal antibodies, be any of the monoclonal antibodies Good.
  • the antibody against the 2 2 receptor protein used in the present invention can be produced according to a known antibody or antiserum production method using the 2 2 receptor protein as an antigen.
  • the P 2 Y 2 receptor protein used in the present invention is administered to a mammal at a site where antibody production is possible by administration, together with a carrier or a diluent.
  • a carrier or a diluent In order to enhance the antibody-producing ability upon administration, complete Freund adjuvant or incomplete Freund adjuvant may be administered. Administration is usually once every 2 to 6 weeks, for a total of 2 to 10 times. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, hidges, and goats, but mouse rats are preferably used.
  • the temperature of the immunized antigen Select individuals with antibody titers from blood animals such as mice, collect spleen or lymph nodes 2-5 days after the last immunization, and fuse antibody-producing cells contained in them with myeloma cells
  • a monoclonal antibody-producing hybridoma can be prepared.
  • the antibody titer in the antiserum can be measured, for example, by reacting the labeled receptor protein described below with the antiserum and then measuring the activity of the labeling agent bound to the antibody.
  • the fusion operation can be carried out according to known methods, for example, the method of Kohler and Milstein [Nature, 2 56 6, pp. 4 95 (1 97 5)].
  • the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but preferably PEG is used.
  • myeloma cells examples include NS-1, P 3 U 1 and SP 2/0, and P 3 U 1 is preferably used.
  • the preferred ratio between the number of antibody producing cells (spleen cells) and the number of myeloma cells is about 1: 1 to 20 : 1, and PEG (preferably P EG 100 00 to PEG 60 00) Is added at a concentration of about 10 to 80%, and cell fusion is carried out efficiently by incubating at about 20 to 4 ° C, preferably about 30 to 37 ° C for about 1 to 10 minutes. it can.
  • a solid phase eg, a microplate
  • a receptor protein antigen is adsorbed directly or with a carrier
  • Add the supernatant and then add anti-immunoglobulin antibody labeled with a radioactive substance or enzyme (if the cells used for cell fusion are mice, use anti-mouse immunoglobulin antibody) or protein A.
  • RPMI 16 40 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum :!
  • a GIT medium (Wako Pure Chemical Industries, Ltd.) containing ⁇ 10% fetal bovine serum or a serum-free medium for culturing Hypridoma (SFM-101, Nissui Pharmaceutical Co., Ltd.) can be used.
  • the culture temperature is usually 20 to 40 ° C, preferably about 37 ° C.
  • the culture time is usually 5 days to 3 weeks, preferably 1 to 2 weeks.
  • Culture can usually be performed under 5% carbon dioxide.
  • the antibody titer of the hybridoma culture supernatant can be measured in the same manner as the antibody titer in the antiserum described above.
  • Monoclonal antibodies can be separated and purified by immunoglobulin separation and purification methods (eg, salting-out method, alcohol precipitation method, isoelectric precipitation method, electrophoresis method, ion exchanger).
  • immunoglobulin separation and purification methods eg, salting-out method, alcohol precipitation method, isoelectric precipitation method, electrophoresis method, ion exchanger.
  • DEAE adsorption / desorption method, ultracentrifugation method, gel filtration method, antigen binding solid phase or active adsorbent such as protein A or protein G
  • the polyclonal antibody of the present invention can be produced by a known method or a method analogous thereto. For example, a complex of an immunizing antigen (P 2 Y 2 receptor protein antigen) and a carrier protein is prepared, and a mammal is immunized in the same manner as in the production of the monoclonal antibody described above. It can be produced by collecting an antibody-containing substance against the ⁇ 2 ⁇ 2 receptor protein used in the invention and separating and purifying the antibody.
  • P 2 Y 2 receptor protein antigen an immunizing antigen
  • carrier protein a mammal is immunized in the same manner as in the production of the monoclonal antibody described above. It can be produced by collecting an antibody-containing substance against the ⁇ 2 ⁇ 2 receptor protein used in the invention and separating and purifying the antibody.
  • the type of carrier protein and the mixing ratio of carrier and hapten are the antibodies against the hapten immunized by cross-linking the carrier.
  • any ratio can be cross-linked as long as it can be efficiently processed.
  • rosin serum albumin ushisailogrobulin, keyhole limpet, mosianin, etc.
  • a method is used in which the hapten 1 is coupled at a ratio of about 0.1 to 20 and preferably about 1 to 5.
  • various condensing agents can be used for hapten and carrier force printing, such as active aldehyde reagent containing aldehyde aldehyde, maleimide active ester, thiol group, and dithiobilidyl group. Used.
  • the condensation product is administered to a warm-blooded animal either at the site where antibody production is possible, or with a carrier or diluent.
  • a carrier or diluent In order to enhance the antibody-producing ability upon administration, complete Freund's adjuvant or incomplete Freund's adjuvant may be administered. Administration can be performed about once every 2 to 6 weeks, usually about 3 to 10 times in total.
  • Polyclonal antibodies can be collected from blood, ascites, etc. of mammals immunized by the above method, preferably blood.
  • the polyclonal antibody titer in the antiserum can be measured in the same manner as the above-described measurement of the antibody titer in the serum.
  • the separation and purification of the polyclonal antibody can be performed according to the same method for separating and purifying the immunoglobulin as the above-described separation and purification of the monoclonal antibody.
  • [rho 2 Upsilon 2 receptor protein C-terminal peptide or antibody that recognizes a salt thereof for example, [rho 2 Upsilon 2 receptor protein C-terminal peptide or antibody that recognizes a salt thereof (in particular, monoclonal Antibody).
  • the 2 2 receptor protein used in the present invention is highly expressed particularly in skeletal muscle.
  • the DNA encoding the P 2 Y 2 'receptor protein used in the present invention can be used as a medicine such as ⁇ or a hypoglycemic agent.
  • D Nyuarufa encoding [rho 2 Upsilon 2 receptor protein used in the present invention are, for example, diabetes mellitus, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic Retinopathy, hyperlipidemia, obesity, metabolic syndrome, sexual dysfunction, skin disease, arthropathy, osteopenia, arteriosclerosis, blood plug disease, dyspepsia, memory learning disorder, etc. preferably used for diabetes be able to. Diabetes includes insulin-dependent (type I) diabetes and non-insulin-dependent (type II) diabetes.
  • the DN 2 encoding the 2 2 receptor protein used in the present invention can be formulated according to conventional means.
  • DNA encoding the P 2 Y 2 receptor protein when used as the above-mentioned hypoglycemic agent, the DNA alone or retrovirus vector, adenovirus vector, adenovirus associated virus vector After inserting into an appropriate vector such as, it can be carried out according to conventional means.
  • the DNA can be used as it is or with an auxiliary agent for promoting intake. Therefore, it can be administered.
  • DNA encoding the P 2 Y 2 receptor protein used in the present invention is orally administered as a tablet, capsule, elixir, mic mouth capsule, etc. It can be used parenterally in the form of sterile solutions with water or other pharmaceutically acceptable liquids or injections such as suspensions.
  • known carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. that are physiologically recognized as D 2 ⁇ ⁇ ⁇ encoding the ⁇ 2 ⁇ 2 receptor protein used in the present invention
  • D 2 ⁇ ⁇ ⁇ encoding the ⁇ 2 ⁇ 2 receptor protein used in the present invention can be prepared by mixing in the unit dosage forms required for accepted formulation practice.
  • the amount of active ingredient in these preparations is such that an appropriate volume within the indicated range can be obtained.
  • Additives that can be incorporated into tablets, force-pellants, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin
  • binders such as gelatin, corn starch, tragacanth, gum arabic
  • excipients such as crystalline cellulose, corn starch, gelatin
  • a swelling agent such as alginic acid, a lubricant such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharine, a flavoring agent such as peppermint, squid mono oil or tea.
  • a liquid carrier such as fats and oils can be further contained in the above type of material.
  • Sterile compositions for injection should be treated according to normal formulation practices such as dissolving or suspending active substances in vehicles such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil, etc. Can do.
  • aqueous solution for injection for example, physiological saline, isotonic solutions containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) are used.
  • Soluble solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80 TM, HCO- 50) You can use it together.
  • the oily liquid for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.
  • solubilizing agents such as benzyl benzoate and benzyl alcohol.
  • the hypoglycemic agent include, for example, a buffer (for example, phosphate buffer, sodium acetate buffer), a soothing agent (for example, benzalkonium chloride, hydrochloride pro-in), and a stabilizer (for example, , Human serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the preparations obtained in this way are safe and have low toxicity.
  • human mammals eg rats, mice, rabbits, hidges, pigs, tusks, cats, dogs, monkeys, etc.
  • the dose of DNA encoding the P 2 Y 2 receptor protein used in the present invention varies depending on the administration target, target organ, symptoms, administration method, etc. In the case of 60 kg, it is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day.
  • the single dose varies depending on the subject of administration, target organ, symptom, administration method, etc.
  • the single dose varies depending on the subject of administration, target organ, symptom, administration method, etc.
  • the single dose varies depending on the subject of administration, target organ, symptom, administration method, etc.
  • the single dose varies depending on the subject of administration, target organ, symptom, administration method, etc.
  • the single dose varies depending on the subject of administration, target organ, symptom, administration method, etc.
  • about 0.1 to 30 mg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to: LO mg is administered by intravenous injection. It is convenient to do.
  • an amount converted per 60 kg can
  • P 2 Y 2 receptor protein or its partial base peptide used in the present invention Anchisensuporinukureochidoma for DN Alpha encoding [rho 2 Upsilon 2 receptor protein used in the present invention for the P 2 Y 2 receptor protein
  • the antibody can be used as a medicine such as a clot raising agent.
  • [rho 2 Upsilon when there are excessive patients of 2 receptor protein a) ⁇ 2 ⁇ 2 antibody to the receptor protein itself or P 2 Y 2 receptor protein is administered to the patient the [rho 2 Upsilon 2 receptor B) an antisense polynucleotide against DN ⁇ ⁇ ⁇ that encodes the P 2 Y 2 receptor protein.
  • the amount of P 2 Y 2 receptor protein in the patient's body is decreased, and receptor function is normalized. can do.
  • antisense polynucleotides against ⁇ 2 ⁇ 2 receptor protein or partial peptides, DN ⁇ ⁇ ⁇ encoding ⁇ 2 ⁇ 2 receptor protein, or antibodies to P 2 Y 2 receptor protein are safe and low toxic. It is useful as a blood glucose raising agent.
  • the present invention is used in [rho 2 Upsilon 2 receptor protein or its parts partial peptide, antisense poly quinuclidine Reochi de or P for DN Alpha encoding
  • [rho 2 Upsilon 2 receptor protein used in the present invention An antibody against 2 Y 2 receptor protein can be used, for example, for hypoglycemia.
  • the DNA alone, retrovirus vector, adenovirus vector, adenovirus association is used as the above-mentioned blood glucose-elevating agent.
  • DNA can be administered as is or with an adjunct to facilitate uptake through a catheter, such as a gene gun or a high-mouth gel catheter.
  • P 2 Y 2 antibody to the receptor protein antisense polynucleotide or P 2 Y 2 receptor protein to DN Alpha encoding are tablets which may be sugar coated as necessary, capsules, elixirs, microcapsules Le It can be used orally as an agent, or parenterally in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or an injection such as a suspension.
  • a known carrier, flavoring agent, and enhancer that is physiologically recognized as an antisense polynucleotide for D 2 ⁇ ⁇ that encodes a 2 2 receptor protein or an antibody for P 2 2 receptor protein used in the present invention.
  • Generally accepted with form, vehicle, preservatives, stabilizers, binders, etc. Can be made by mixing in the unit dosage form required for the implementation of the formulated product. The amount of active ingredient in these preparations is such that an appropriate volume within the indicated range can be obtained.
  • Additives that can be mixed into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, For example, a swelling agent such as gelatin or alginic acid, a lubricant such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharin, a flavoring agent such as peppermint, cocoa oil or chili is used.
  • a liquid carrier such as fats and oils can be further contained in the above type of material.
  • Sterile compositions for injection should be treated according to normal pharmaceutical practice, such as dissolving or suspending active substances in vehicles such as water for injection, naturally produced vegetable oils such as sesame oil and coconut oil. Can do.
  • aqueous solution for injection for example, physiological saline, isotonic solutions containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) are used.
  • Dissolving aids for example, alcohol (eg, ethanol), polyalcohol (eg, propylene dallicol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80 TM , HCO- 50) Do it anyway.
  • oily liquid for example, sesame oil, soybean oil and the like are used, and they may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.
  • solubilizing agents such as benzyl benzoate and benzyl alcohol.
  • the blood glucose-elevating agent include a buffer (for example, phosphate buffer, sodium acetate buffer), a soothing agent (for example, benzalkonium chloride, hydrochloride pro-in), and a stabilizer (for example, , Human serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the preparations obtained in this way are safe and low toxic.
  • humans such as mammals (eg rat, mouse, rabbit, hidge, pig, ushi, Cats, dogs, monkeys, etc.).
  • Antisense polynucleotide against DN ⁇ ⁇ ⁇ encoding P 2 Y 2 receptor protein used in the present invention The dose varies depending on the administration subject, target organ, symptom, administration method, etc.
  • administration for example, in hypoglycemic patients (as 60 kg), generally about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1. 0 to 20 mg.
  • the single dose varies depending on the subject of administration, target organ, symptom, administration method, etc.
  • hypoglycemic patients 60 kg
  • about 0.1 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day is administered by intravenous injection. Is convenient. In the case of other animals, an amount converted per 6 O kg can be administered.
  • the dose of the antibody against the P 2 Y 2 receptor protein used in the present invention varies depending on the administration subject, target organ, symptom, administration method, etc., but in the case of oral administration, generally, for example, hypoglycemia In a patient (as 60 kg), it is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day.
  • the single dose varies depending on the subject of administration, target organ, symptoms, administration method, etc.
  • a hypoglycemic patient 60 (in kg)
  • about 0.1 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg is administered intravenously per day. Is convenient.
  • an amount converted per 60 kg can be administered.
  • the dose of the P 2 Y 2 receptor protein used in the present invention varies depending on the administration subject, target organ, symptom, administration method, and the like.
  • patients with hypoglycemia (As 0 kg) is about 0.1 to 1: OO mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day.
  • dosing once The amount varies depending on the administration subject, target organ, symptom, administration method, etc.
  • it is usually about 0.00 per day in a hypoglycemic patient (as 60 kg).
  • an amount converted per 60 kg can be administered.
  • the DN ⁇ ⁇ ⁇ encoding the P 2 Y 2 receptor protein used in the present invention can be used as a prop for humans or mammals (eg rat, mouse, rabbit, hidge, pig, ushi, Cats, dogs, monkeys, etc.) can detect an abnormality (gene abnormality) in DNA or mRNA encoding the P 2 Y 2 receptor protein or its partial peptide used in the present invention. It is useful as a genetic diagnostic agent for damage, mutation or reduction of expression of DNA or mRNA, increase of DNA or mRNA, or excessive expression.
  • mammals eg rat, mouse, rabbit, hidge, pig, ushi, Cats, dogs, monkeys, etc.
  • the above-described genetic diagnosis using the DNA encoding the P 2 Y 2 receptor protein used in the present invention is, for example, the known Northern hybridization using the PCR-SSCP method (Genomics, No. 1). 5 ⁇ , 8 7 4-8 7 9 (1 9 8 9), Proceedings of the National Academy, Op The National, Power Demi 1 Ob, Sciences 'Ob' USA of Sciences of the United States oi America;, pp. 86, pp. 2 706-2 770 (1 9 8 9)).
  • the P 2 Y 2 receptor protein used in the present invention when a decrease in the expression of the P 2 Y 2 receptor protein used in the present invention is detected by Northern hyperpridition, for example, it is related to dysfunction of the ⁇ 2 ⁇ 2 receptor protein used in the present invention. It can be diagnosed that the disease is highly likely or is likely to be affected in the future. In addition, when overexpression of the ⁇ 2 ⁇ 2 receptor protein used in the present invention is detected by Northern hyperprecipitation, for example, it is used in the present invention. It can be diagnosed that the disease is likely caused by overexpression of the P 2 Y 2 receptor protein, or is likely to be affected in the future.
  • diseases associated with dysfunction of ⁇ 2 ⁇ 2 receptor protein used in the present invention include diabetes, impaired glucose tolerance, keto-cissis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, Examples include hyperlipidemia, obesity, metabolic syndrome, sexual dysfunction, skin disease, arthropathy, osteopenia, arteriosclerosis, thrombotic disease, dyspepsia, memory learning disorder.
  • diseases caused by overexpression of [rho 2 Upsilon 2 receptor proteins are found used in the present invention include, for example, hypoglycemia.
  • the antibodies of the present invention is capable of specifically recognizing the [rho 2 Upsilon 2 receptor protein, quantification of [rho 2 Upsilon 2 receptor protein in a test fluid, especially, for quantification by Sanditsuchi immunoassay can do.
  • the antibody of the present invention is competitively reacted with a test solution and a labeled P 2 Y 2 receptor protein, and the labeled ⁇ 2 ⁇ 2 receptor protein bound to the antibody is A method for quantifying ⁇ 2 ⁇ 2 receptor protein in a test solution, characterized by measuring a ratio;
  • one antibody is an antibody that recognizes the heel end of the P 2 Y 2 receptor protein and the other antibody is an antibody that reacts with the C terminus of the ⁇ 2 ⁇ 2 receptor protein. It is desirable that
  • the [rho 2 Upsilon 2 can perform quantification of [rho 2 Upsilon 2 receptor protein with monoclonal antibodies to the receptor protein Ho force tissue staining or the like.
  • the antibody molecule itself may be used, or the F (ab ′) 2 , Fab, or Fab fraction of the antibody molecule may be used.
  • the method for quantifying the P 2 Y 2 receptor protein using the antibody of the present invention is not particularly limited, and an antibody corresponding to the amount of antigen in the solution to be measured (for example, ⁇ 2 ⁇ 2 receptor protein mass).
  • any method can be used as long as the amount of antigen or antibody-antigen complex is detected by chemical or physical means, and this is calculated from a standard curve prepared using a standard solution containing a known amount of antigen.
  • the measurement method described above may be used.
  • a nephrometry, a competition method, an immunometric method and a sandwich method are preferably used, but the sandwich method described below is particularly preferable in terms of sensitivity and specificity.
  • Examples of the labeling agent used in the measurement method using the labeling substance include radioisotopes, enzymes, fluorescent substances, and luminescent substances.
  • radioisotope for example, [ 125 I], [ 131 I], [ 3 H], [ 14 C] and the like are used.
  • enzyme a stable enzyme having a large specific activity is preferable.
  • / 3-galactosidase, _darcosidase, alkaline phosphatase, peroxidase, phosphonate dehydrogenase and the like are used.
  • As the fluorescent material for example, fluorescamine, fluorescein isothiocyanate and the like are used.
  • luminescent substance for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used.
  • a piotin-avidin system can be used for the binding between the antibody or antigen and the labeling agent.
  • ⁇ ⁇ 2 ⁇ 2 receptor protein when a decrease in the concentration of 2 2 ⁇ 2 receptor protein is detected by quantifying the concentration of P 2 ⁇ Y 2 receptor protein using the antibody of the present invention, for example, ⁇ ⁇ 2 ⁇ 2 receptor It can be diagnosed that the disease is related to dysfunction of body protein, or is likely to be affected in the future. Further, when increasing concentrations of [rho 2 Upsilon 2 receptor protein is detected, for example, is a disease caused by overexpression of [rho 2 Upsilon 2 receptor protein, or likely to suffer future Can be diagnosed.
  • the diseases associated with dysfunction of the ⁇ 2 ⁇ 2 receptor protein of the present invention include: diabetes, glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy Hyperlipidemia, obesity, metabolic syndrome, sexual function Disorders, skin diseases, arthropathy, osteopenia, arteriosclerosis, thrombotic diseases, dyspepsia, memory learning disorders, etc.
  • diseases caused by overexpression of the P 2 Y 2 receptor protein of the present invention include hypoglycemia.
  • test compound is administered at the same time as the drug or physical stress, and after a certain period of time after administration (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 2 4 hours later), the amount of mRNA in the P 2 Y 2 receptor protein contained in the cells can be quantified and analyzed.
  • the test compound is mixed in the medium and cultured for a certain period of time (after 1 day to 7 days, preferably after 1 day to 3 days, more preferably after 2 days to 3 days later), it can be carried out by quantifying and analyzing the amount of mRN of the P 2 Y 2 receptor protein contained in the transformant.
  • the compound obtained by using the screening method of the present invention or a salt thereof is a compound having an action of changing the expression level of the P 2 Y 2 receptor protein used in the present invention.
  • (I) [rho 2 Upsilon 2 receptor protein, [rho 2 Upsilon mediated cell stimulating activities 2 receptor protein (e.g., Arakidon acid release, Asechirukorin release, intracellular C a 2+ release, cell Activity that promotes or suppresses intracellular cAMP generation, intracellular cGMP generation, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH reduction, etc.
  • the compound that changes the expression level of the P 2 Y 2 receptor protein of the present invention obtained by the above screening method can be used for a disease associated with dysfunction of ⁇ 2 ⁇ 2 receptor protein. .
  • the expression level of [rho 2 Upsilon 2 receptor protein of the present invention is increased, a compound that enhances the cell stimulating activity, against diseases associated with functional insufficiency of [rho 2 Upsilon 2 receptor protein of the present invention It is useful as a hypoglycemic agent.
  • the expression level of [rho 2 Upsilon 2 receptor protein of the present invention reduces, compounds that decrease the cell-stimulating activity is safe and low toxic for diseases caused by overexpression of [rho 2 Upsilon 2 receptor protein of the present invention It is useful as a blood glucose raising agent.
  • the diseases associated with dysfunction of the [rho 2 Upsilon 2 receptor protein of the present invention diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, high Examples include lipemia, obesity, metabolic syndrome, sexual dysfunction, skin disease, arthropathy, osteopenia, arteriosclerosis, thrombotic disease, dyspepsia, memory learning disorder.
  • diseases caused by overexpression of [rho 2 Upsilon 2 receptor protein of the present invention include, for example, hypoglycemia.
  • salts of the compounds include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metals such as sodium and potassium; alkaline earth metals such as calcium), etc. And physiologically acceptable acid addition salts are particularly preferred.
  • physiologically acceptable acid addition salts are particularly preferred.
  • examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid). Salts with acid, succinic acid, tartaric acid, citrate, malic acid, succinic acid, benzoic acid, methane sulfonic acid, benzene sulfonic acid, etc.).
  • the compound obtained by using the screening method of the present invention or a salt thereof is used as a pharmaceutical composition
  • it can be formulated according to conventional means.
  • the compound may be used orally as tablets, capsules, elixirs, microcapsules, etc. with sugar coating as required, or with water or other pharmaceutically acceptable liquids.
  • It can be used parenterally in the form of an injectable solution such as a sterile solution or suspension.
  • injectable solution such as a sterile solution or suspension.
  • the amount of active ingredient in these preparations is such that an appropriate volume within the indicated range is obtained.
  • additives examples include gelatin, corn starch, tragacanth, binders such as gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid.
  • a leavening agent such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharin, a flavoring agent such as peppermint, cocoa oil, or cherry is used.
  • a liquid carrier such as fats and oils can be further contained in the above type of material.
  • Sterile compositions for injection should be treated according to normal formulation practices such as dissolving or suspending active substances in vehicles such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil, etc. Can do.
  • aqueous solution for injection for example, physiological saline, isotonic solutions containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) are used.
  • Suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene dallicol), nonionic surfactants (eg, polysorbate 80 TM , HCO-50), etc. You may use together.
  • the oily liquid examples include sesame oil and soybean oil, which may be used in combination with a solubilizing agent such as benzyl benzoate or benzyl alcohol.
  • the pharmaceutical composition includes, for example, a buffer (for example, a phosphate buffer, Sodium acetate buffer), soothing agent (eg, benzalkonium chloride, hydrochloride pro-in, etc.), stabilizer (eg, human serum albumin, polyethylene glycol, etc.), preserved IJ (eg, benzyl alcohol, phenol) Etc.), and may be blended with an antioxidant.
  • a buffer for example, a phosphate buffer, Sodium acetate buffer
  • soothing agent eg, benzalkonium chloride, hydrochloride pro-in, etc.
  • stabilizer eg, human serum albumin, polyethylene glycol, etc.
  • preserved IJ eg, benzyl alcohol, phenol
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the preparations obtained in this way are safe and low toxic.
  • they can be used in human mammals (eg rats, mice, rabbits, hidges, pigs, tusks, cats, dogs, monkeys, etc.). Can be administered.
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method, etc., but in the case of oral administration, for example, in diabetic patients (as 6 O kg), About 0. per day! ⁇ 10 O mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.
  • the single dose varies depending on the subject of administration, subject organ, symptoms, administration method, etc.
  • diabetic patients for example, diabetic patients (as 60 kg) , About 0.1 to 30 mg per day, preferably about 0.1:! To 20 mg, more preferably about 0.1 to 10 mg is administered by intravenous injection. Is convenient. In the case of other animals, an amount converted per 60 kg can be administered.
  • an appropriate ⁇ 2 ⁇ 2 receptor protein fraction and a label are used.
  • Ligand is needed.
  • a labeled ligand As the labeled ligand, a labeled ligand, a labeled ligand analog compound, or the like is used. For example [3 H], [125 I], [14 C], [35 S] A ligand labeled with such as is used.
  • the 3 ⁇ 4 specifically, in performing the disk re one Eng compounds that alter the binding property between a ligand and P 2 Y 2 receptor protein, cells or cell containing first [rho 2 Upsilon 2 receptor protein membrane Prepare ⁇ 2 ⁇ 2 receptor protein preparation by suspending aliquots in a buffer suitable for screening.
  • any buffer can be used as long as it does not inhibit the binding of ⁇ 2 2 receptor protein such as ⁇ ⁇ 4 to 10 (preferably ⁇ ⁇ 6 to 8) phosphate buffer or Tris monohydrochloride buffer. But you can.
  • surfactants such as CHAP S, Tween -80 TM (Kaoichi Atlas), digitonin, and deoxycholate can be added to the buffer.
  • protease inhibitors such as PMS F, leupeptin, E-64 (manufactured by Peptide Institute) and pepstatin can be added for the purpose of suppressing degradation of receptors and ligands by protease.
  • a fixed amount (5 0 00 cpm to 5 OOOOO cpm) of labeled ligand is added to 0.01 ml to 10 ml of the receptor solution, and simultaneously 1 0— 4 M to 1 0_ 1Q M test compound Coexist.
  • the reaction is carried out at about 0 to 50 ° C., preferably about 4 to 37 ° C. for about 20 minutes to 24 hours, preferably about 30 minutes to 3 hours.
  • Another method for screening for a compound that changes the binding property between the ligand and the P 2 Y 2 receptor protein is, for example, cell stimulating activity via the ⁇ 2 ⁇ 2 receptor protein (eg, arachidon). Acid release, acetylcholine release, intracellular Ca2 + release, intracellular cAMP generation, intracellular cGMP generation, wild boar Tall phosphate production, cell membrane potential fluctuation, phosphorylation of intracellular protein, — fos activation, activity to promote or suppress pH reduction, etc. Especially, intracellular Ca 2+ concentration increasing activity, intracellular c The measurement is carried out using a known method or a commercially available measurement kit.
  • ⁇ 2 ⁇ 2 receptor protein eg, arachidon.
  • cells containing the P 2 Y 2 receptor protein of the present invention are cultured on a multiwall plate or the like. Before performing screening, replace with a fresh medium or an appropriate buffer that is not toxic to cells, add test compounds, etc., and incubate for a certain period of time. Quantify the resulting product according to the respective method. If it is difficult to produce a substance (for example, C 2+ , c AMP, etc.) that serves as an index of cell stimulating activity using a degrading enzyme contained in the cell, an inhibitor for the degrading enzyme is added to perform the assay. It's okay. Further, the activity such as cAMP production inhibition can be detected as a production inhibitory action on cells whose basic production amount has been increased with forskolin or the like.
  • a substance for example, C 2+ , c AMP, etc.
  • cells expressing an appropriate P 2 Y 2 receptor protein are required.
  • the cells expressing [rho 2 Upsilon 2 receptor protein, cells having a native-type [rho 2 Upsilon 2 receptor protein strains, such as the above recombinant [rho 2 Upsilon 2 cell lines expressing the receptor protein is desirable.
  • test compounds include peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, and animal tissue extracts. These compounds are novel compounds. It may be a known compound.
  • [rho 2 Upsilon 2 receptor based on the protein atomic positions coordinate and ligand binding pocket of the active site of, compounds I designed to bind to the ligand binding pocket is preferably used.
  • Measurement of [rho 2 Upsilon 2 receptor protein located atomic coordinates and ligand binding pocket Bok of the active site of the leaves in be conducted using a known method or a method analogous thereto.
  • the specific evaluation method of whether the compound that changes the binding property between the ligand and the P 2 Y 2 receptor protein of the present invention is an agonist or an antagonist is the following (i) or (ii) Just follow.
  • test compound is contacted with a cell containing the P 2 Y 2 receptor protein of the present invention, and the above-mentioned cell stimulating activity is measured.
  • Test compounds that have the cell-stimulating activity is Agonisu preparative for [rho 2 Upsilon 2 receptor protein.
  • P 2 Y 2 receptor protein compounds that activate e.g., ligand
  • activate e.g., ligand
  • the [rho 2 Upsilon 2 receptor protein compounds and the test compounds was measured cell stimulating activity when contacted with a cell containing a [rho 2 Upsilon 2 receptor protein and compared.
  • [Rho 2 Upsilon 2 receptor protein test compound capable of decline the cell stimulating activity induced by the compound that activates the are antagonistic preparative for [rho 2 Upsilon 2 receptor protein.
  • Examples of the screening kit of the present invention include the following.
  • CHO cells expressing the [rho 2 Upsilon 2 receptor protein used in the present invention and passaged 1 2 well plates at 5 X 1 0 5 cells / well, 3 7. C, 5% C 0 2 , 95% air cultured for 2 days.
  • the compound obtained by using the screening method or the screening kit of the present invention or a salt thereof is a compound having an action of changing the binding property between the ligand and the P 2 Y 2 receptor protein.
  • a compound having a cell stimulating activity via a G protein-coupled receptor so-called agonist for ⁇ 2 ⁇ 2 receptor protein
  • mouth a compound not having the cell stimulating activity (so-called, antagonists g) for [rho 2 Upsilon 2 receptor proteins, binding force between (c) a compound that potentiates the binding affinity of the ligand and the [rho 2 Upsilon 2 receptor protein, or
  • ligands and [rho 2 Upsilon 2 receptor protein Use a compound that reduces.
  • Examples of the compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, and the like. These compounds may be novel compounds or known compounds.
  • the compounds may be filed with compounds designed based on the position of the atomic coordinates Oyopi ligand binding Boke' bets of the active site of the above-mentioned [rho 2 Upsilon 2 receptor protein.
  • Compound or its enhance binding force between Agonisu bets or ligand and [rho 2 Upsilon 2 receptor protein for disk re one Jung method or subscription one obtained using a Jung for kit
  • [rho 2 Upsilon 2 receptor protein of the present invention Salts, for example, diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, obesity, metabolic syndrome, sexual dysfunction, skin diseases, joints It can be used for diseases such as symptom, osteopenia, arteriosclerosis, thrombotic disease, indigestion, memory learning disorder, etc., and is preferably useful as a hypoglycemic agent for diabetes.
  • antagonists bets or Li ligand and [rho 2 Upsilon respect is [rho 2 Upsilon 2 receptor protein obtained using the disk Li one Jung method or kit for screening of the present invention.
  • a compound or its compound that decreases the binding power to the receptor protein This salt is useful, for example, as a blood glucose raising agent.
  • the compound obtained by using the screening method or the screening kit of the present invention or a salt thereof is a compound or a salt thereof that changes the expression level of the P 2 Y 2 receptor protein.
  • Drugs such as other diabetes treatment agents, diabetic complication treatment agents, hyperlipidemia treatment agents, antihypertensive agents, anti-obesity agents, diuretics, chemotherapeutic agents, immunotherapy agents (hereinafter abbreviated as combination drugs) Can be used in combination.
  • the administration time of the compound obtained by using the screening method or the screening kit of the present invention or a salt thereof and a concomitant drug is not limited, and these are administered simultaneously to the administration subject. May be administered after a time lag.
  • the dose of the concomitant drug can be appropriately selected based on the clinically used dose. Further, the compounding ratio of the compound obtained by using the screening method or screening kit of the present invention or a salt thereof and a concomitant drug should be appropriately selected depending on the administration subject, administration route, target disease, symptom, combination, etc. Can do. For example, when the administration subject is a human, to Agonisu sheet 1 part by weight In example embodiment, a combination drug 0.0 may Re Re for 1 to 1 0 0 parts by Q
  • the compound obtained by using the screening method or screening kit of the present invention or a salt thereof is used as the above pharmaceutical composition, it can be formulated according to conventional means.
  • the compound is sterilized orally as tablets, capsules, elixirs, microcapsules or the like with sugar coating as required, or with water or other pharmaceutically acceptable liquids. It can be used parenterally in the form of injections such as solutions or suspensions.
  • the compound in a unit dosage form generally required for practicing recognized formulations along with known physiologically recognized carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. It can be produced by mixing. The amount of active ingredient in these preparations should be such that an appropriate volume within the indicated range is obtained. Additives that can be mixed into glazes, force-pellants, etc.
  • binders such as gelatin, corn starch, tragacanth, arabic gum, excipients such as crystalline cellulose, corn starch , Gelatinizing agents such as alginic acid, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharine, flavoring agents such as peppermint, cocoa oil or tea Used.
  • a liquid carrier such as fats and oils can be further contained in the above type of material.
  • Sterile compositions for injection should be treated according to normal pharmaceutical practice, such as dissolving or suspending active substances in vehicles such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil, etc. be able to.
  • aqueous solution for injection for example, physiological saline, isotonic solutions containing buducose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) are used.
  • Suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80 TM, HCO—50) You may use together.
  • alcohol eg, ethanol
  • polyalcohol eg, propylene glycol, polyethylene glycol
  • nonionic surfactants eg, polysorbate 80 TM, HCO—50
  • the above pharmaceutical composition includes, for example, a buffer (for example, phosphate buffer, sodium acetate buffer), a soothing agent (for example, benzalkonium chloride, hydrochloride pro-in, etc.), a stabilizer ( For example, human serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc. may be added.
  • a buffer for example, phosphate buffer, sodium acetate buffer
  • a soothing agent for example, benzalkonium chloride, hydrochloride pro-in, etc.
  • a stabilizer for example, human serum albumin, polyethylene glycol, etc.
  • preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants etc.
  • the preparations obtained in this way are safe and low toxic.
  • they can be used in human mammals (eg rats, mice, rabbits, hidges, pigs, tusks, cats, dogs, monkeys, etc.). Can be administered.
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method, and the like.
  • sick patients about 0.1 to 10 O mg per day, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.
  • the single dose varies depending on the subject of administration, subject organ, symptom, administration method, etc.
  • the single dose varies depending on the subject of administration, subject organ, symptom, administration method, etc.
  • the single dose varies depending on the subject of administration, subject organ, symptom, administration method, etc.
  • an injection for example, diabetic patients (as 6 O kg )
  • About 0.1 to 30 mg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg is administered by intravenous injection. Is convenient.
  • an amount converted per 60 kg can be administered.
  • mRN A Messenger Ribonucleic acid
  • EDTA ethylenediamine tetraacetic acid
  • SDS sodium dodecyl sulfate
  • TC thiazolidine-4 (R) monocarboxamide group
  • substituents, protecting groups and reagents frequently used in the present specification are described below. Expressed with a number.
  • the amino acid sequence of the human P 2 Y 2 receptor protein used in the present invention is shown.
  • FIG. 2 shows the base sequence of cDNA encoding the human 2-2 receptor protein used in the present invention.
  • [SEQ ID NO: 6] Shows the base sequence of c D ⁇ ⁇ ⁇ ⁇ encoding rat P 2 Y 2 .
  • Example 2 shows the base sequence of the upstream primer used in Example 2 for amplifying the human P 2 Y 2 region.
  • Example 2 shows the base sequence of the downstream primer used in Example 2 for amplifying the human 2 2 region.
  • Example 2 Used in Example 2, shows the base sequence of the probe for detecting amplification of human [rho 2 Upsilon 2 region. '
  • Example 2 shows the base sequence of an upstream primer used in Example 2 for amplifying the mouse 2 2 region.
  • Example 2 shows the base sequence of the downstream primer used in Example 2 for amplifying the mouse 2 2 region.
  • Example 2 shows the base sequence of a probe used in Example 2 for detecting amplification of the mouse 2 2 region.
  • the base sequence of the upstream primer used in Example 4 for amplifying the rat 2 2 region is shown.
  • Example 4 The base sequence of the downstream primer used in Example 4 for amplifying the rat 2 2 region is shown. [SEQ ID NO: 15] 'This shows the base sequence of the probe used in Example 4 for detecting the amplification of the rat P 2 Y 2 region.
  • Example 1 The present invention will be described in more detail with reference to examples below, but these do not limit the scope of the present invention.
  • Example 1
  • IH ygro (+) [Invitrogen; # V 8 7 0 2 0] is a transfer reagent, Lipofectamine 2 0 0 0 (Invitrogen was used according to the attached protocol) L 6 — SG 4— 8 1 Introduced into 3 7 ° (:, After 4 hours at 5% C_ ⁇ 2 conditions, 1 washed with the medium used during the seeding and 1 2 hour incubation at 3 7 ° C, 5% C_ ⁇ 2 conditions. After washing once with Mimim Essential Essential Medium containing 0.1% fatty acidfree BSA (Sigma) 3 7 in this medium. C and 5% C 0 2 conditions were cultured for 4 hours.
  • the TaqMan method is used to belong to a family of target G protein-coupled receptors, tyrosine kinases, ion channels, etc. in any human and mouse RNA sample.
  • the presence of gene-derived mRNA and the production amount thereof were quantified, and the expression levels of genes belonging to the family, such as target G protein-coupled receptor, tyrosine phosphorylase receptor, and ion channel, were analyzed.
  • target GPCR genes include ORL; Ml; M2; M3; M4; M5; Al; A2A; A2B; A3; A1A; aIB; aID; a2A; a2B; a2C; ⁇ 1; j32 3; ATI; AT2; BB1; BB2; bb3; Bl; B2; CBl; CB2; CCRl; CCR2; CCR3; CCR4; CCR5; CCR6; CCR7; CCR8; CCR9; CCR10; CXCRl; CXCR2; CXCR3; CXCR4; CXCR5 CX3CR1; XCRl; C3a; C5a; fMLP; CCKl; CCK2; CRF1; CRF2; Dl; D2; D3; D4; D5; ETA; ETB; GAL1; GAL2; GAL3; mglul; mglu2;
  • Amplification was performed using the following samples.
  • RNA derived from adult normal skeletal muscle (C 1 ontech) was purchased.
  • the mouse sample was C 5 7 B LZ 6 right femoral skeletal muscle removed,
  • Total I NA was prepared according to the manual of I sogen (Nitsubon Gene).
  • I sogen Neitsubon Gene
  • a serially diluted solution of synthesized cDNA of the amplification region was used.
  • the amplification of the region of human P 2 Y 2 mRN was performed using the upstream primer (SEQ ID NO: 7) and the downstream primer (SEQ ID NO: 8). Detection was performed using a probe (SEQ ID NO: 9) (Applied Biosystems Japan Co., Ltd.).
  • Amplification of the mouse P 2 Y 2 mRNA region was performed using an upstream primer (SEQ ID NO: 10) and a downstream primer (SEQ ID NO: 11). Detection was performed using a probe (SEQ ID NO: 1 2) (Applied Biosystems Japan Co., Ltd.).
  • the probe In order to enable detection in the TaqMan format, the probe has a 5 'end with a fluorescein-type fluorescent dye (FAM: reporter) and a 3' end with a rhodamine-type fluorescent dye (TAMRA: It was labeled with Quenchia. When the labeled probe was not hybridized, the fluorescence of the reporter was suppressed due to the movement of fluorescence resonance energy. In order to prevent extension of the probe by DNA polymerase during amplification, the 3 'end of the probe was synthesized with a phosphate block.
  • FAM fluorescein-type fluorescent dye
  • TAMRA rhodamine-type fluorescent dye
  • reverse transcription reaction was performed on 40 ⁇ g of the RNA described above using random primers.
  • Use reverse transcriptase Superscript II (Invitrogen) react according to the attached protocol, precipitate with ethanol, and add 4 ⁇ / l TE (10 mM Tris—HC 1, 1 mM) Dissolved in EDTA pH 8.0).
  • the synthesized cDNA was dissolved in TE and diluted to 10 ng 1 in TE containing 50 ⁇ g / m 1 yeast tRNA.
  • diluted cDNA solution 2.5 ⁇ 1 (equivalent to 25 ng RNA), TaqMan (registered trademark) Universal PCR Master Mix (Applied Biosystems Japan Co., Ltd.) was used as the amplification reaction reagent. The total amount of the reaction solution was adjusted. Each The final concentration of lymer and probe was in accordance with the manual.
  • TaqMan (registered trademark) PCR was performed with the ABI PRISM (registered trademark) 7900HT Sequence Detection System (Applied Biosystems Japan Co., Ltd.), and the temperature cycle used was: TaqMan (registered trademark). Universal PCR Master Mix (Applied) DBiosystems Japan) manual.
  • 7900HT SDS software Applied Biosystems Japan Co., Ltd. was used, and the number of copies per sample was calculated by the absolute quantification method.
  • the amount of gene mRNA belonging to the family such as other target G protein-coupled receptors, tyrosin oxidase type receptors, and ion channels was quantified. Then, by comparing the production of all target G protein-coupled receptors, tyrosine phosphatase receptors, ion channels, and other ⁇ -transduced mRNAs, normal human and mouse normal skeletons are compared. Each target G protein-coupled receptor, tyrosine phosphatase receptor, ion channel, and other gene expression levels in muscle were analyzed.
  • FIG. 1 shows the analysis results.
  • RNAs derived from various human tissues were purchased.
  • each tissue was extracted from C 5 7 BL 6 forces, and total RNA was prepared according to the manual of I sogen (Nitsubon Gene).
  • I sogen Nicoon Gene
  • c For DNA synthesis, 40 ⁇ g of RNA was subjected to reverse transcription using random primers. The reverse transcriptase Super Script II (Invitrogen) was used, reacted according to the attached protocol, ethanol precipitated and dissolved in 400 ⁇ I ⁇ . The synthesized cD ⁇ ⁇ was dissolved in ⁇ ⁇ and diluted to 10 ng / ⁇ 1 in TE containing 50 ⁇ g Zm 1 yeast tRNA.
  • TaqMan (registered trademark) PCR was performed with ABI PRISM (registered trademark) 7900HT Sequence Detection System (Applied Biosystems Japan Co., Ltd.), and the temperature cycle used was TaqMan (registered trademark) Universal PCR Master Mix (Applied Biotechnology). System Japan Co., Ltd.) manual.
  • TaqMan analysis of amplified products the number of copies per sample was calculated by absolute quantification using 7900HT SDS software (Applied Biosystems Japan Co., Ltd.).
  • Both cells of mouse skeletal muscle cell line C 2 C 12 (AT CC) and rat skeletal muscle cell line L 6 (distributed from JC Lawrence Jr) are 10% FBS ⁇ lnvitrogen, 50 units / mL Penicillin G Sodium, 5
  • the cells were cultured in Dulbecco's Modified Eagle Medium (Invitrogen) containing 0 g / m1 Streptomycin sulfate (Invitrogen).
  • C 2 C 12 was differentiated by replacing it with Dulbe ⁇ Sco, s Modified Eagle Medium containing 2% horse serum (ICN).
  • L 6 promoted differentiation by replacing 2% FBS with-Minimum Essential Medium Invitrogen.
  • RNAs were prepared according to the manual of I sogen (Nitsubon Gene). .
  • the primers and probes used for quantification are the same as those used in Example 1 for mice, and the upstream primers (SEQ ID NO: 13) and downstream primers (SEQ ID NO: 14) are used for rats. It was. Detection was performed using a probe (sequence number: 15). The final concentration of each primer and probe was in accordance with Magyuannole.
  • TaqMan (registered trademark) PCR was performed with ABI PRISM (registered trademark) 7900HT Sequence Detection System (Applied Biosystems Japan Co., Ltd.), and the temperature cycle used was TaqMan (registered trademark) Universal PCR Master Mix (Applied Dubai). System Japan Co., Ltd.) Quantitative TaqMan analysis of amplification products was tested by the absolute quantification method using 7900HT SDS software (Applied Biosystems Japan Co., Ltd.). The number of copies per fee was calculated.
  • L 6 cells (L 6— SG 4— 8 1 1) are 10% FBS (I ⁇ Vitogen), 50 units / mL Penicillin G Sodium, 50 ⁇ g / m 1 Streptomycin sulfate ( Invitrogen) and cultivated in Dulbecco's Modified Eagle Medium (Invitrogen).
  • P 2 Y 2 agonists can be a novel drug target with a wide range of hypoglycemic effects in diabetics with both insulin secretion deficiency and insulin resistance.

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Abstract

La présente invention a pour objet un agent de régulation de glycémie qui stimule ou inhibe l'apport de glucose à l’aide du récepteur P2Y2 dans le muscle squelettique, afin de régler la glycémie sanguine.
PCT/JP2005/021068 2004-11-11 2005-11-10 Agent de régulation de glycémie WO2006052007A1 (fr)

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US9161562B2 (en) 2004-06-04 2015-10-20 Horizon Science Pty Ltd Natural sweetener
US9364016B2 (en) 2006-09-19 2016-06-14 The Product Makers (Australia) Pty Ltd Extracts derived from sugar cane and a process for their manufacture
US9572852B2 (en) 2011-02-08 2017-02-21 The Product Makers (Australia) Pty Ltd Sugar extracts
US10350259B2 (en) 2013-08-16 2019-07-16 The Product Makers (Australia) Pty Ltd Sugar cane derived extracts and methods of treatment
US10614684B2 (en) 2017-01-31 2020-04-07 The United States Government As Represented By The Treatment of kidney diseases associated with elevated AVP
US11730178B2 (en) 2012-08-28 2023-08-22 Poly Gain Pte Ltd Extraction method

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9161562B2 (en) 2004-06-04 2015-10-20 Horizon Science Pty Ltd Natural sweetener
US9364016B2 (en) 2006-09-19 2016-06-14 The Product Makers (Australia) Pty Ltd Extracts derived from sugar cane and a process for their manufacture
US10226502B2 (en) 2011-02-08 2019-03-12 The Product Makers (Australia) Pty Ltd Sugar extract
US9572852B2 (en) 2011-02-08 2017-02-21 The Product Makers (Australia) Pty Ltd Sugar extracts
US9717771B2 (en) 2011-02-08 2017-08-01 The Product Makers (Australia) Pty Ltd Sugar extract
US11730178B2 (en) 2012-08-28 2023-08-22 Poly Gain Pte Ltd Extraction method
US10024846B2 (en) * 2012-10-25 2018-07-17 The United States Of America As Represented By The Department Of Veterans Affairs Methods for treating diet-induced obesity by decreasing or inhibiting P2Y2 purinergic receptor expression or activity
US10107795B2 (en) * 2012-10-25 2018-10-23 The United States Of America As Represented By The Department Of Veterans Affairs Composition and methods for the prevention and treatment of diet-induced obesity
US20150259692A1 (en) * 2012-10-25 2015-09-17 Department Of Veterans Affairs Composition and methods for the prevention and treatment of diet-induced obesity
US20160377598A1 (en) * 2012-10-25 2016-12-29 Bellamkonda K. Kishore Composition and methods for the prevention and treatment of diet-induced obesity
US10350259B2 (en) 2013-08-16 2019-07-16 The Product Makers (Australia) Pty Ltd Sugar cane derived extracts and methods of treatment
US10614684B2 (en) 2017-01-31 2020-04-07 The United States Government As Represented By The Treatment of kidney diseases associated with elevated AVP
US11354990B2 (en) 2017-01-31 2022-06-07 The United States Government As Represented By The Department Of Veterans Affairs Treatment of kidney diseases associated with elevated AVP

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